Mutual antagonism of mouse-adaptation mutations in HA and PA proteins on H9N2 virus replication.

Avian H9N2 viruses have wide host range among the influenza A viruses. However, knowledge of H9N2 mammalian adaptation is limited. To explore the molecular basis of the adaptation to mammals, we performed serial lung passaging of the H9N2 strain A/chicken/Hunan/8.27 YYGK3W3-OC/2018 (3W3) in mice and identified six mutations in the hemagglutinin (HA) and polymerase acidic (PA) proteins. Mutations L226Q, T511I, and A528V of HA were responsible for enhanced pathogenicity and viral replication in mice; notably, HA-L226Q was the key determinant. Mutations T97I, I545V, and S594G of PA contributed to enhanced polymerase activity in mammalian cells and increased viral replication levels in vitro and in vivo. PA-T97I increased viral polymerase activity by accelerating the viral polymerase complex assembly. Our findings revealed that the viral replication was affected by the presence of PA-97I and/or PA-545V in combination with a triple-point HA mutation. Furthermore, the double- and triple-point PA mutations demonstrated antagonistic effect on viral replication when combined with HA-226Q. Notably, any combination of PA mutations, along with double-point HA mutations, resulted in antagonistic effect on viral replication. We also observed antagonism in viral replication between PA-545V and PA-97I, as well as between HA-528V and PA-545V. Our findings demonstrated that several antagonistic mutations in HA and PA proteins affect viral replication, which may contribute to the H9N2 virus adaptation to mice and mammalian cells. These findings can potentially contribute to the monitoring of H9N2 field strains for assessing their potential risk in mammals.

Highly Pathogenic Avian Influenza A (H5N1) Virus in Swans, Central China, 2021.

Six highly pathogenic avian influenza (HPAI) H5N1 viruses (clade 2.3.4.4b) were detected in migratory birds in Hubei Province in November 2021. Phylogenetic analysis indicated that the viruses in the study included two different reassortants between H5N1 viruses that were circulating in Eurasia and low-pathogenic avian influenza viruses (LPAIVs). Several amino acid substitutions that contributed to the enhanced replication or virulence in mammals were observed in these viruses, suggesting a potential threat of the H5N1 viruses to human health. IMPORTANCE Here, we obtained the whole-genomes of six H5N1 viruses from dead or rescued wild birds in Hubei Province. These viruses were divided into two genotypes and had different evolutionary trajectories from previously reported H5N1 viruses in China. Extensive reassortment events between high-pathogenic (HP) and low-pathogenic (LP) avian influenza viruses (AIVs) were observed in these viruses. Moreover, a key amino acid analysis also suggests a potential threat of H5N1 viruses to public health. Our work explored the prevalent patterns of H5N1 viruses in wild birds and replenished the viral population data of H5N1 viruses in central China.

Genetic and Pathogenic Characterization of Avian Influenza Virus in Migratory Birds between 2015 and 2019 in Central China.

Active surveillance of avian influenza virus (AIV) in wetlands and lakes is important for exploring the gene pool in wild birds. Through active surveillance from 2015 through 2019, 10,900 samples from wild birds in central China were collected, and 89 AIVs were isolated, including 2 subtypes of highly pathogenic AIV and 12 of low-pathogenic AIV; H9N2 and H6Ny were the dominant subtypes. Phylogenetic analysis of the isolates demonstrated that extensive intersubtype reassortments and frequent intercontinental gene exchange occurred in AIVs. AIV gene segments persistently circulated in several migration seasons, but interseasonal persistence of the whole genome was rare. The whole genomes of one H6N6 and polymerase basic 2 (PB2), polymerase acidic (PA), hemagglutinin (HA), neuraminidase (NA), M, and nonstructural (NS) genes of one H9N2 virus were found to be of poultry origin, suggesting a spillover of AIVs from poultry to wild birds. Importantly, one H9N2 virus only bound to human-type receptor, and one H1N1, four H6, and seven H9N2 viruses possessed dual receptor-binding capacity. Nineteen of 20 representative viruses tested could replicate in the lungs of mice without preadaptation, which poses a clear threat of infection in humans. Together, our study highlights the need for intensive AIV surveillance. IMPORTANCE Influenza virus surveillance in wild birds plays an important role in the early recognition and control of the virus. However, the AIV gene pool in wild birds in central China along the East Asian-Australasian flyway has not been well studied. Here, we conducted a 5-year AIV active surveillance in this region. Our data revealed the long-term circulation and prevalence of AIVs in wild birds in central China, and we observed that intercontinental gene exchange of AIVs is more frequent and continuous than previously thought. Spillover events from poultry to wild bird were observed in H6 and H9 viruses. In addition, in 20 representative viruses, 12 viruses could bind human-type receptors, and 19 viruses could replicate in mice without preadaption. Our work highlights the potential threat of wild bird AIVs to public health.

Dominant subtype switch in avian influenza viruses during 2016-2019 in China.

We have surveyed avian influenza virus (AIV) genomes from live poultry markets within China since 2014. Here we present a total of 16,091 samples that were collected from May 2016 to February 2019 in 23 provinces and municipalities in China. We identify 2048 AIV-positive samples and perform next generation sequencing. AIV-positive rates (12.73%) from samples had decreased substantially since 2016, compared to that during 2014-2016 (26.90%). Additionally, H9N2 has replaced H5N6 and H7N9 as the dominant AIV subtype in both chickens and ducks. Notably, novel reassortants and variants continually emerged and disseminated in avian populations, including H7N3, H9N9, H9N6 and H5N6 variants. Importantly, almost all of the H9 AIVs and many H7N9 and H6N2 strains prefer human-type receptors, posing an increased risk for human infections. In summary, our nation-wide surveillance highlights substantial changes in the circulation of AIVs since 2016, which greatly impacts the prevention and control of AIVs in China and worldwide.